Stabilized antibody-containing formulations

ABSTRACT

The present invention relates to antibody-containing lyophilized formulations free from reducing sugars, non-reducing sugars, sugar alcohols or polysaccharides as excipients and including one or more amino acid selected from the group consisting of arginine, histidine, lysine, serine, proline, glycine, alanine and threonine or a salt thereof.

TECHNICAL FIELD

The present invention relates to antibody-containing formulations,especially stable high-concentration lyophilized antibody formulations.

BACKGROUND ART

With the development of genetic recombination technology, variousprotein formulations became supplied in stable amounts. To ensurestability, these formulations are supplied in the dosage form of alyophilized protein ingredient powder to be dissolved just before use ina separately packaged water-soluble diluent or in the dosage form of aprotein solution formulation containing additives for improvingstability.

Recently, various antibody formulations have been developed and appliedfor practical use, but many of the antibody formulations are used asformulations for intravenous injection. However, there are increasingdemands in the medical field for developing antibody-containingformulations in the form of self-injectable formulations forsubcutaneous injection.

In designing antibody-containing formulations for subcutaneousinjection, the antibody concentration in the solution to be administeredmust be high because the injectable volume by subcutaneous injection isnormally limited despite of a single high antibody dose (about 100-200mg). Thus, there are demands for developing high-concentrationantibody-containing formulations by means of lyophilization-basedconcentration.

JPA No. 2004-532798/WO 2002/030463/U.S. Pat. No. 6,875,432 discloses aconcentrated protein formulation with decreased viscosity including asalt and/or a buffer, but describes only the effect of reducing theviscosity of the solution and nothing about stability.

JPA No. 2004-538287/WO 2003/009817 discloses a lyophilizedpharmaceutical formulation prepared by lyophilizing an aqueouspreparation containing an IgG antibody at a high concentration,Polysorbate, sucrose, and optionally serine and/or mannitol in ahistidine buffer (pH about 5.5 to about 6.5). However, this applicationfocuses on the stabilizing effect of sucrose, but describes nothingabout the stabilizing effect by adding serine and histidine.

High-concentration antibody-containing solutions tend to formhigh-viscosity solutions by the properties of proteins as macromoleculesand molecular interactions. Moreover, they are normally lyophilized inthe presence of large amounts of lyoprotectants such as sugars duringthe preparation of lyophilized formulations to maintain their cakingproperties and stability. However, sugars enhance molecular interactionsto increase viscosity, and the resulting high-viscosity formulations aredifficult to dispense, draw into syringes and subcutaneously inject.Thus, novel methods for preparing stable lyophilized formulationswithout adding sugars are needed.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a stable antibodyformulation that forms little dimers or low molecular weight degradationproducts of the antibody during the process of preparing ahigh-concentration antibody-containing formulation by means oflyophilization-based concentration without adding sugars and duringstorage and reconstitution of the resulting high-concentrationlyophilized formulation.

Means of Solving the Problems

As a result of careful studies to attain the above object, weaccomplished the present invention on the basis of the finding thathigh-concentration antibody-containing lyophilized formulations can beobtained without adding sugars as fillers by adding one or more aminoacid selected from the group consisting of arginine, histidine, lysine,serine, proline, glycine, alanine and threonine or a salt thereof.

Accordingly, the present invention provides the following:

-   -   (1) An antibody-containing lyophilized formulation free from        reducing sugars, non-reducing sugars, sugar alcohols or        polysaccharides as fillers and including one or more amino acid        selected from the group consisting of arginine, histidine,        lysine, serine, proline, glycine, alanine and threonine or a        salt thereof.    -   (2) The formulation as defined in (1) wherein the antibody        concentration of the lyophilized formulation after        reconstitution is 10 mg/mL or more.    -   (3) The formulation as defined in (2) wherein the antibody        concentration of the lyophilized formulation after        reconstitution is 50 mg/mL or more.    -   (4) The formulation as defined in (3) wherein the antibody        concentration of the lyophilized formulation after        reconstitution is 100 mg/mL or more.    -   (5) The formulation as defined in any one of (1) to (4) wherein        the content of the amino acid or a salt thereof is 270 moles or        more per mole of the antibody.    -   (6) The formulation as defined in (5) wherein the content of the        amino acid or a salt thereof is 380 moles or more per mole of        the antibody.    -   (7) The formulation as defined in (6) wherein the content of the        amino acid or a salt thereof is 540 moles or more per mole of        the antibody.    -   (8) The formulation as defined in any one of (1) to (7) wherein        the pH of the solution after reconstitution is 4-8.    -   (9) The formulation as defined in (8) wherein the pH of the        solution after reconstitution is 5.0-7.5.    -   (10) The formulation as defined in any one of (1) to (9) wherein        the antibody is a chimeric antibody, humanized antibody or human        antibody.    -   (11) The formulation as defined in (10) wherein the antibody is        an anti-IL-6 receptor antibody.    -   (12) The formulation as defined in any one of (1) to (11)        wherein the amino acid or a salt thereof is one or more amino        acid selected from the group consisting of arginine, histidine        and lysine or a salt thereof.    -   (13) The formulation as defined in (12) wherein the amino acid        or a salt thereof is arginine or a salt thereof.    -   (14) The formulation as defined in any one of (1) to (13)        wherein the viscosity after reconstitution is 20 mPa·s or less.    -   (15) The formulation as defined in (14) wherein the viscosity        after reconstitution is 15 mPa·s or less.    -   (16) The formulation as defined in (15) wherein the viscosity        after reconstitution is 12 mPa·s or less.    -   (17) An antibody-containing lyophilized formulation including        one or more amino acid selected from the group consisting of        arginine, histidine, lysine, serine, proline, glycine, alanine        and threonine or a salt thereof as a lyoprotectant or a filler        characterized in that it includes 25 mg/ml or more of the amino        acid or a salt thereof when the antibody concentration of the        lyophilized formulation is 20 mg/mL or more, or it includes 12.5        mg/ml or more of the amino acid or a salt thereof when the        antibody concentration is 30 mg/mL or more, or it includes 6.25        mg/ml or more of the amino acid or a salt thereof when the        antibody concentration is 40 mg/mL or more.    -   (18) The formulation as defined in (17) characterized in that it        includes 25 mg/ml or more of the amino acid or a salt thereof        when the antibody concentration of the lyophilized formulation        is 30 mg/mL or more, or it includes 12.5 mg/ml or more of the        amino acid or a salt thereof when the antibody concentration is        40 mg/mL or more.    -   (19) A process for preparing an antibody-containing lyophilized        formulation including one or more amino acid selected from the        group consisting of arginine, histidine, lysine, serine,        proline, glycine, alanine and threonine or a salt thereof as a        lyoprotectant or a filler, comprising the step of lyophilizing a        solution before lyophilization having a total concentration of        reducing sugars, non-reducing sugars, sugar alcohols and        polysaccharides of less than 20 mg/mL.    -   (20) An antibody-containing lyophilized formulation including        one or more amino acid selected from the group consisting of        argienie, lysine, serine, serine, proline, glycine, alanine and        threonine or a salt thereof as a lyoprotectant or a filler,        wherein the ratio per vial of the total weight of reducing        sugars, non-reducing sugars, sugar alcohols and polysaccharides        to the antibody weight is less than 0.5:1.    -   (21) A protein-containing lyophilized formulation free from        reducing sugars, non-reducing sugars, sugar alcohols or        polysaccharides as fillers and including one or more amino acid        selected from the group consisting of arginine, histidine,        lysine, serine, proline, glycine, alanine and threonine or a        salt thereof.    -   (22) A process for preparing a protein-containing lyophilized        formulation including one or more amino acid selected from the        group consisting of arginine, histidine, lysine, serine,        proline, glycine, alanine and threonine or a salt thereof as a        lyoprotectant or a filler, comprising the step of lyophilizing a        solution before lyophilization having a total concentration of        reducing sugars, non-reducing sugars, sugar alcohols and        polysaccharides of less than 20 mg/mL.    -   (23) A protein-containing lyophilized formulation including one        or more amino acid selected from the group consisting of        arginine, histidine, lysine, serine, proline, glycine, alanine        and threonine or a salt thereof as a lyoprotectant or a filler,        wherein the ratio per vial of protein weight to the total weight        of reducing sugars, non-reducing sugars, sugar alcohols and        polysaccharides is less than 1:0.5.

THE MOST PREFERRED EMBODIMENTS OF THE INVENTION

Antibodies used in the methods of the present invention are notspecifically limited so far as they bind to a desired antigen, and maybe polyclonal or monoclonal, but preferably monoclonal becausehomogeneous antibodies can be stably produced.

Monoclonal antibodies used in the present invention include not onlymonoclonal antibodies derived from animals such as humans, mice, rats,hamsters, rabbits, sheep, camels and monkeys but also artificiallymodified recombinant antibodies such as chimeric antibodies, humanizedantibodies and bispecific antibodies. The antibodies may belong to anyimmunoglobulin class including, but not limited to, IgG such as IgG1,IgG2, IgG3 or IgG4, and IgA, IgD, IgE and IgM, preferably IgG and IgM.

Moreover, the antibodies of the present invention include not only wholeantibodies, but also antibody fragments such as Fv, Fab and F(ab)₂ orfragmented antibodies such as monovalent or multivalent single chain Fvs(scFv, sc(Fv)₂ and diabodies such as scFv dimers) in which the variableregions of an antibody are joined together via a linker such as apeptide linker.

The antibodies of the present invention described above can be preparedby processes well known to those skilled in the art.

Hybridomas producing monoclonal antibodies can be basically constructedby known techniques as follows. That is, a desired antigen or a cellexpressing a desired antigen is used as an immunizing antigen toimmunize host cells according to a standard immunization technique, andthe resulting immunized cells are fused to known parent cells by astandard cell fusion technique, and then the fused cells are screenedfor monoclonal antibody-producing cells (hybridomas) by a standardscreening method. Construction of hybridomas can be performed accordingto the method of e.g. Milstein et al. (Kohler. G. and Milstein, C.,Methods Enzymol. (1981) 73: 3-46). If the antigen has lowimmunogenicity, it can be bound to an immunogenic macromolecule such asalbumin and used for immunization.

Recombinant antibodies can be used, which are produced by transforming ahost with a suitable vector containing an antibody gene cloned from ahybridoma using genetic engineering techniques (e.g., see Carl, A. K.Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES,Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).Specifically, the cDNA sequences for the variable regions (V regions) ofan antibody are synthesized from mRNA of a hybridoma using a reversetranscriptase. Thus obtained DNA sequences encoding the V regions of thedesired antibody are linked to the DNA sequences encoding the constantregions (C regions) of the desired antibody and integrated into anexpression vector. Alternatively, the DNA sequences encoding the Vregions of the antibody can be integrated into an expression vectorcontaining the DNA sequences for the C regions of the antibody. They areintegrated into the expression vector in such a manner that they can beexpressed under the control of regulatory regions such as enhancers andpromoters. Then, a host cell can be transformed with this expressionvector to express the antibody.

In the present invention, recombinant antibodies, i.e. antibodiesartificially modified to reduce antigenicity in humans or to attainother purposes, such as chimeric antibodies and humanized antibodies canbe used. These modified antibodies can be prepared by known processes.Chimeric antibodies consist of the heavy and light chain variableregions of an antibody from a non-human mammal such as a mouse and theheavy and light chain constant regions of a human antibody and can beobtained by linking the DNA sequences encoding the variable regions ofthe mouse antibody to the DNA sequences for the constant regions of thehuman antibody and transforming a host with an expression vectorcontaining the linked sequences to allow it to produce a chimericantibody.

Humanized antibodies are also called reshaped human antibodies andobtained by grafting the complementarity-determining regions (CDRs) ofan antibody from a non-human mammal such as a mouse into thecomplementarity-determining regions of a human antibody and typical generecombination techniques for preparing them are also known.Specifically, DNA sequences designed to link the CDRs of a mouseantibody to the framework regions (FRs) of a human antibody aresynthesized by PCR from several oligonucleotides prepared to haveterminal overlapping regions. The resulting DNA sequences are linked tothe DNA sequences encoding the constant regions of the human antibodyand then integrated into an expression vector, which is transformed intoa host to allow it to produce a reshaped antibody (see European PatentPublication No. EP 239400, International Publication No. WO 96/02576).The FRs of the human antibody linked by the CDRs are selected in such amanner that the complementarity-determining regions form an appropriateantigen-binding site. If necessary, reshaped human antibodies may havesome amino acid changes in the framework regions of the variable regionsso that the complementarity-determining regions form an appropriateantigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

Methods for obtaining human antibodies are also known. For example, adesired human antibody having a binding activity for a desired antigencan be obtained by in vitro immunizing human lymphocytes with thedesired antigen or a cell expressing the desired antigen and fusing theimmunized lymphocytes to human myeloma cells such as U266 (see JPB No.HEI-1-59878). A desired human antibody can also be obtained byimmunizing a transgenic animal having all human antibody generepertoires with an antigen (see International Publications Nos. WO93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO96/33735). Methods for obtaining a human antibody by panning using ahuman antibody library are also known. For example, phages binding to anantigen can be selected by expressing the variable regions of a humanantibody as single chain antibody fragments (scFv) on phage surfaces bya phage display method. The DNA sequences encoding the variable regionsof the human antibody binding to the antigen can be determined byanalyzing the genes of the selected phages. A whole human antibody canbe obtained by preparing a suitable expression vector containing thedetermined DNA sequences of the scFv fragments binding to the antigen.These methods have already been well known from WO 92/01047, WO92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO95/15388.

When an antibody is to be prepared by transforming a preliminarilyisolated antibody gene into a suitable host, a combination of thesuitable host with an expression vector can be used. Suitable eukaryoticcells for use as hosts include animal, plant and fungal cells. Knownanimal cells include (1) mammal cells such as CHO, COS, myeloma, BHK(baby hamster kidney), HeLa and Vero cells; (2) amphibian cells such asXenopus oocytes; or (3) insect sells such as sf9, sf21 and Tn5. Knownplant cells include cells of Nicotiana such as Nicotiana tabacum, whichcan be used as callus cultures. Known fungi include yeasts such asSaccharomyces spp., e.g. Saccharomyces serevisiae and filamentous fungisuch as Aspergillus spp., e.g. Aspergillus niger. Prokaryotic cells canbe used as producing systems using bacterial cells. Known bacterialcells include E. coli and Bacillus subtilis. Antibodies can be obtainedby transforming these cells with an antibody gene of interest andculturing the transformed cells in vitro.

Moreover, the antibodies of the present invention may be antibodyfragments or fragmented antibodies and modified antibodies. For example,antibody fragments or fragmented antibodies include Fab, (Fab′)₂, Fv, orsingle chain Fvs (scFv, sc(Fv)₂, etc.) in which the heavy and lightchain Fv fragments are joined via a suitable linker (Huston, J. S. etal., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). Specifically,an antibody is treated with an enzyme such as papain or pepsin toproduce antibody fragments or the genes encoding these antibodyfragments are constructed and introduced into an expression vector andthen expressed in a suitable host cell (e.g., see Co, M. S. et al., J.Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., MethodsEnzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., MethodsEnzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121,652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669;Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).

Modified antibodies including antibodies conjugated with variousmolecules such as polyethylene glycol (PEG) can also be used. Thesemodified antibodies are also included in the “antibodies” of the presentinvention. Such modified antibodies can be obtained by chemicallymodifying the antibodies produced as above. These methods have alreadybeen established in this field of art.

Antibodies contained in the formulations of the present inventioninclude, but not limited to, anti-tissue factor antibodies, anti-IL-6receptor antibodies, anti-HM1.24 antigen monoclonal antibodies,anti-parathyroid hormone related peptide antibodies (anti-PTHrPantibodies), anti-glypican-3 antibodies, anti-ganglioside GM3antibodies, anti-TPO receptor agonist antibodies, alternative antibodiesto coagulation VIII factor, etc.

Preferred reshaped humanized antibodies for use in the present inventioninclude humanized anti-IL-6 receptor antibodies (hPM-1) (seeInternational Publication No. WO92-19759), humanized anti-HM1.24 antigenmonoclonal antibodies (see International Publication No. WO98-14580),humanized anti-parathyroid hormone related peptide antibodies(anti-PTHrP antibodies) (see International Publication No. WO98-13388),humanized anti-tissue factor antibodies (see International PublicationNo. WO99-51743), and humanized anti-glypican-3 IgG1κ antibodies (seeInternational Application No. PCT/JP05/013103). Especially preferredhumanized antibodies for use in the present invention are humanizedanti-IL-6 receptor antibodies.

Preferred human IgM antibodies include recombinant human IgManti-ganglioside GM3 antibodies (see International Publication No.WO05-05636).

Preferred fragmented antibodies include anti-TPO receptor diabodies (seeInternational Publication No. WO02-33072) and anti-CD47 agonistdiabodies (see International Publication No. WO01-66737).

The antibody-containing lyophilized formulations of the presentinvention preferably have an antibody concentration of 10 mg/mL or more,more preferably 50 mg/mL or more, still more preferably 80 mg/mL ormore, especially 100 mg/mL or more after the lyophilized formulationsare reconstituted.

The antibody-containing lyophilized formulations of the presentinvention may have any antibody concentration before lyophilization.However, when a high-concentration solution formulation is prepared byreconstituting a lyophilized formulation with a smaller volume of waterthan contained in the solution before lyophilization, i.e. when theso-called lyophilization-based concentration technique is applied, theantibody concentration before lyophilization is preferably 1 mg/mL ormore, more preferably 10 mg/mL or more, especially 20 mg/mL or more. Theconcentration factor of the antibodies in the lyophilization-basedconcentration technique is preferably 2- to 50-fold, more preferably 2-to 20-fold, especially 2- to 6-fold.

The amino acid added as a stabilizer to the formulations of the presentinvention is an amino acid selected from the group consisting ofarginine, histidine, lysine, serine, proline, glycine, alanine andthreonine and salts thereof, preferably an amino acid selected from thegroup consisting of L-arginine, L-histidine, L-lysine, L-serine,L-proline, glycine, L-alanine and L-threonine and salts thereof. Anespecially preferred amino acid is L-arginine or a salt thereof.

The content of the amino acid in the formulations of the presentinvention is preferably 270 moles or more per mole of the antibody, morepreferably 380 moles or more per mole of the antibody, still morepreferably 540 moles or more per mole of the antibody.

In the high-concentration antibody-containing lyophilized formulationsof the present invention, the pH of the solution after reconstitution ofthe lyophilized formulations is preferably 4-8, more preferably 5.0-7.5,still more preferably 5.5-7.2.

In the formulations of the present invention, the viscosity afterreconstitution is preferably 20 mPa·s or less, more preferably 15 mPa·sor less, still more preferably 12 mPa·s or less.

The formulations of the present invention are characterized in that theyare free from reducing sugars, non-reducing sugars, sugar alcohols orpolysaccharides as fillers. Normally, when reducing sugars, non-reducingsugars, sugar alcohols or polysaccharides are added toprotein-containing formulations as fillers for lyophilized formulations,they should be included at a level of 20 mg/mL or more in a solutionbefore lyophilization. Thus, the expression “free from reducing sugars,non-reducing sugars, sugar alcohols or polysaccharides as fillers” asused herein means that the total concentration of reducing sugars,non-reducing sugars, sugar alcohols or polysaccharides included in asolution before lyophilization during the preparation of aprotein-containing lyophilized formulation is less than 20 mg/mL,preferably 10 mg/mL or less, more preferably 5 mg/mL or less, still morepreferably 1 mg/mL or less, especially substantially zero.

The present invention also provides an antibody-containing lyophilizedformulation including one or more amino acid selected from the groupconsisting of arginine, histidine, serine, proline, glycine, alanine andthreonine or a salt thereof as a lyoprotectant or a filler, wherein theratio per vial of the total weight of reducing sugars , non-reducingsugars, sugar alcohols and polysaccharide to the antibody weight is lessthan 0.5:1, The ratio per vial of the total weight of reducing sugars,non-reducing sugars, sugar alcohols and polysaccharides to the antibodyweight is preferably less than 0.5:1, more preferably 0.25:1 or less,still more preferably 0.125:1 or less, still more preferably 0.125:1 orless, and most preferably the formulation free from reducing sugars,non-reducing sugars, sugar alcohols and polysaccharides.

As used herein, the reducing sugars include glucose, fructose, maltose,lactose; the non-reducing sugars include sucrose, trehalose, raffinose,neotrehalose; the sugar alcohols include mannitol, sorbitol, maltitol,erythritol; and the polysaccharides include dextran, cyclodextrins(α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin), carboxymethylcellulose, hydroxypropylmethyl cellulose.

The formulations of the present invention can form stable antibodyformulations that ensure good caking and form little dimers or lowmolecular weight degradation products of the antibody during thelyophilization process and during storage and reconstitution of thelyophilized formulations without adding reducing sugars, non-reducingsugars, sugar alcohols or polysaccharides as fillers. Moreover,easy-to-use lyophilized formulations can be obtained in which theincrease of the viscosity of the high-concentration antibody-containingsolution after reconstitution can be prevented because they containremarkably smaller amounts of sugars as compared with conventionallyophilized formulations or they contain substantially no sugars.

Preferably, the formulations of the present invention substantiallyconsist of the following components:

-   -   A) an antibody,    -   B) one or more amino acid selected from the group consisting of        arginine, histidine, lysine, serine, proline, glycine, alanine,        threonine and salts thereof,    -   C) a salt as a buffer, and    -   D) a surfactant.

“Substantially consist of” means that the formulations do not containany component other than the optional additives described below ascommonly contained in formulations such as suspending agents,solubilizers, isotonizing agents, preservatives, adsorption inhibitors,diluents, fillers, pH modifiers, soothing agents, sulfur-containingreducing agents, antioxidants, etc.

When a formulation of the present invention is to be prepared, an aminoacid selected from the group consisting of arginine, histidine, lysine,serine, proline, glycine, alanine, threonine and salts thereof is addedto a prepared solution containing an antibody before lyophilization andthen the solution is lyophilized. Thus, a good lyophilized cake isformed without using sugars conventionally used as fillers, and at thesame time, the production of dimers of the antibody can be inhibitedduring the lyophilization process.

The present invention also provides an antibody-containing lyophilizedformulation including one or more amino acid selected from the groupconsisting of arginine, histidine, lysine, serine, proline, glycine,alanine and threonine or a salt thereof as a lyoprotectant or a fillercharacterized in that it includes 25 mg/ml or more of the amino acid ora salt thereof when the antibody concentration of the lyophilizedformulation is 20 mg/mL or more, or it includes 12.5 mg/ml or more ofthe amino acid or a salt thereof when the antibody concentration is 30mg/mL or more, or it includes 6.25 mg/ml or more of the amino acid or asalt thereof when the antibody concentration is 40 mg/mL or more.Preferably, it includes 25 mg/ml or more of the amino acid or a saltthereof when the antibody concentration of the lyophilized formulationis 30 mg/mL or more, or it includes 12.5 mg/ml or more of the amino acidor a salt thereof when the antibody concentration is 40 mg/mL or more.

The present invention also provides a process for preparing anantibody-containing lyophilized formulation including one or more aminoacid selected from the group consisting of arginine, histidine, lysine,serine, praline, glycine, alanine and threonine or a salt thereof as alyoprotectant or a filler, comprising the step of lyophilizing asolution before lyophilization having a total concentration of reducingsugars, non-reducing sugars, sugar alcohols and polysaccharides of lessthan 20 mg/mL. The total concentration of reducing sugars, non-reducingsugars, sugar alcohols or polysaccharides is preferably 10 mg/mL orless, more preferably 5 mg/mL or less, still more preferably 1 mg/mL orless, especially substantially zero.

Typical examples of surfactants include:

-   -   nonionic surfactants, e.g., sorbitan fatty acid esters such as        sorbitan monocaprylate, sorbitan monolaurate, sorbitan        monopalmitate; glycerin fatty acid esters such as glycerin        monocaprylate, glycerin monomyristate, glycerin monostearate;        polyglycerin fatty acid esters such as decaglyceryl        monostearate, decaglyceryl distearate, decaglyceryl        monolinoleate; polyoxyethylene sorbitan fatty acid esters such        as polyoxyethylene sorbitan monolaurate, polyoxyethylene        sorbitan monooleate, polyoxyethylene sorbitan monostearate,        polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan        trioleate, polyoxyethylene sorbitan tristearate; polyoxyethylene        sorbitol fatty acid esters such as polyoxyethylene sorbitol        tetrastearate, polyoxyethylene sorbitol tetraoleate;        polyoxyethylene glycerin fatty acid esters such as        polyoxyethylene glyceryl monostearate; polyethylene glycol fatty        acid esters such as polyethylene glycol distearate;        polyoxyethylene alkyl ethers such as polyoxyethylene lauryl        ether; polyoxyethylene polyoxypropylene alkyl ethers such as        polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene        polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene        cetyl ether; polyoxyethylene alkyl phenyl ethers such as        polyoxyethylene nonyl phenyl ether; polyoxyethylene hardened        castor oils such as polyoxyethylene castor oil, polyoxyethylene        hardened castor oil (polyoxyethylene hydrogenated castor oil);        polyoxyethylene beeswax derivatives such as polyoxyethylene        sorbitol beeswax; polyoxyethylene lanolin derivatives such as        polyoxyethylene lanolin; polyoxyethylene fatty acid amides such        as polyoxyethylene stearic acid amide having an HLB of 6-18;    -   anionic surfactants, e.g., alkyl sulfates having a C10-18 alkyl        group such as sodium cetyl sulfate, sodium lauryl sulfate,        sodium oleyl sulfate; polyoxyethylene alkyl ether sulfates        having an average EO mole number of 2-4 and a C10-18 alkyl group        such as sodium polyoxyethylene lauryl sulfate; alkyl        sulfosuccinic acid ester salts having a C8-18 alkyl group such        as sodium laurylsulfosuccinate; and    -   natural surfactants, e.g., lecithin; glycerophospholipids;        sphingophospholipids such as sphingomyelin; sucrose fatty acid        esters of C12-18 fatty acids. Formulations of the present        invention can contain one or more of these surfactants in        combination.

Preferred surfactants are polyoxyethylene sorbitan fatty acid esters andpolyoxyethylene polyoxypropylene alkyl ethers, more preferablyPolysorbates 20, 21, 40, 60, 65, 80, 81, 85 and Pluronic surfactants,most preferably Polysorbates 20 and 80 and Pluronic F-68 (poloxamer188).

The amount of surfactants to be added to the antibody formulations ofthe present invention is typically 0.0001-10% (w/v), preferably0.001-5%, more preferably 0.005-3%.

Salts that can be used as buffers include inorganic salts such as sodiumphosphate, potassium phosphate and sodium bicarbonate; and organic saltssuch as sodium citrate, potassium citrate and sodium acetate. Acids thatcan be used as buffers include phosphoric acid, carbonic acid, citricacid, succinic acid, malic acid, etc. Other buffers that can be usedinclude Tris buffers and Good's buffers such as MES and MOPS.

The formulations of the present invention can contain suspending agents,solubilizers, isotonizing agents, preservatives, adsorption inhibitors,diluents, fillers, pH modifiers, soothing agents, sulfur-containingreducing agents, antioxidants, etc., if desired.

Examples of suspending agents include methylcellulose, Polysorbate 80,hydroxyethylcellulose, gum acacia, tragacanth powder, sodiumcarboxymethylcellulose, polyoxyethylene sorbitan monolaurate, etc.

Solubilizers include polyoxyethylene hydrogenated castor oil,Polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitanmonolaurate, Macrogols, castor oil fatty acid ethyl esters, etc.

Isotonizing agents include e.g., sodium chloride, potassium chloride,calcium chloride, etc.

Preservatives include e.g., methyl paraoxybenzoate, ethylparaoxybenzoate, sorbic acid, phenol, cresol, chlorocresol, etc.

Adsorption inhibitors include e.g., human serum albumin, lecithin,dextran, ethylene oxide/propylene oxide copolymers,hydroxypropylcellulose, methylcellulose, polyoxyethylene hydrogenatedcastor oil, polyethylene glycol, etc.

Sulfur-containing reducing agents include e.g., N-acetylcysteine,N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine,thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodiumthiosulfate, glutathione, and sulfhydryl-containing compounds such asthioalkanoic acid having 1 to 7 carbon atoms.

Antioxidants include e.g., erythorbic acid, dibutylhydroxytoluene,butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acidand salts thereof, L-ascorbyl palmitate, L-ascorbyl stearate, sodiumbisulfite, sodium sulfite, triamyl gallate, propyl gallate or chelatingagents such as disodium ethylenediamine tetraacetate (EDTA), sodiumpyrophosphate and sodium metaphosphate.

The antibody-containing lyophilized formulations of the presentinvention are normally administered via parenteral routes, for exampleby injection (e.g. subcutaneous, intravenous or intramuscular injection)or via percutaneous, mucosal, nasal or pulmonary route, but can also beorally administered. The formulations of the present invention areespecially suitable for subcutaneous injection because the injectablevolume is normally limited despite of a single high antibody dose (about100-200 mg).

As shown from the results of the examples below, the formulations of thepresent invention can provide good lyophilized compositions withoutusing sugars conventionally used as fillers by adding a specific aminoacid, thereby reducing the viscosity of the solution afterreconstitution, which was an issue especially when high-concentrationantibody-containing formulations are prepared by means oflyophilization-based concentration. Moreover, stable antibodyformulations that form little dimers or low molecular weight degradationproducts of the antibody during the lyophilization process and duringstorage and reconstitution of the resulting high-concentrationlyophilized formulations can be obtained.

Although the present invention has been illustrated in terms ofantibodies, which are the most preferred embodiment, the presentinvention is not limited to such antibodies and is also applicable toother proteins. Proteins used in the formulations of the presentinvention include, but not limited to, hematopoietic factors such asgranulocyte colony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), erythropoietin (EPO) andthrombopoietin, cytokines such as interferon, IL-1 and IL-6, as well astissue plasminogen activator (TPA), urokinase, serum albumin, bloodcoagulation factor VIII, leptin, insulin, stem cell factor (SCF), etc.

The following examples further illustrate the present invention without,however, limiting the scope of the invention thereto. Various changesand modifications can be made by those skilled in the art on the basisof the description herein, and such changes and modifications are alsoincluded in the present invention.

EXAMPLES

Antibody Sample

A humanized anti-IL-6 receptor antibody was prepared by the methoddescribed in Reference example 2 of JPA No. HEI-8-99902 using the humanelongation factor Ia promoter described in Example 10 of InternationalPublication No. WO 92/19759. It is sometimes referred to as MRA in thetables in the examples below.

Example 1 Selection of Filler Types Having the Effect of StabilizingLyophilized Formulations

Filler types having the effect of stabilizing lyophilized formulationscontaining the humanized anti-IL-6 receptor antibody were sought withthe aim of stabilizing them before and after the lyophilization processand on storage.

In this study, evaluation samples of sample Nos. 1-11 were prepared toevaluate the effect of adding the following ten fillers. The formulae ofthe prepared solutions of the evaluation samples before lyophilizationare as follows.

TABLE 1 [Formulae of prepared solutions (before lyophilization)] Sam-Filler ple Antibody Content Polysorbate Phosphate No. mg/mL Type (mg/mL)80 mg/mL buffer mM pH 1 40 Sucrose 25 0.5 15 7.0 2 40 Trehalose 25 0.515 7.0 3 40 Raffinose 25 0.5 15 7.0 4 40 Mannitol 25 0.5 15 7.0 5 40Dextran 25 0.5 15 7.0 6 40 Arginine 25 0.5 15 7.0 7 40 Histidine 25 0.515 7.0 8 40 Glycine 25 0.5 15 7.0 9 40 Serine 25 0.5 15 7.0 10 40Proline 25 0.5 15 7.0 11 40 — — 0.5 15 7.0

A glass vial containing 2 mL of the prepared solution of each evaluationsample was lyophilized under the following conditions to give alyophilized formulation.

TABLE 2 Lyophilization conditions Degree of Step Shelf temperaturePeriod vacuum Initial freezing −50° C. ca. 24 hr — Primary drying −20°C. 70 hr 10 Pa  Secondary drying (1)  25° C. 28 hr 6 Pa Secondary drying(2)  30° C. 10 hr 6 Pa

The formulae of the lyophilized formulations of the evaluations samplesare as follows.

TABLE 3 [Formulae of lyophilized formulations (after lyophilization)]Sam- Filler Phosphate ple Antibody Content Polysorbate buffer No.mg/vial Type (mg/vial) 80 mg/vial μmol/vial pH 1 80 Sucrose 50 1 30 7.02 80 Trehalose 50 1 30 7.0 3 80 Raffinose 50 1 30 7.0 4 80 Mannitol 50 130 7.0 5 80 Dextran 50 1 30 7.0 6 80 Arginine 50 1 30 7.0 7 80 Histidine50 1 30 7.0 8 80 Glycine 50 1 30 7.0 9 80 Serine 50 1 30 7.0 10 80Proline 50 1 30 7.0 11 80 — — 1 30 7.0

In order to evaluate stability during the lyophilization process, thepurity of each sample of the prepared solutions before lyophilizationand the lyophilized formulations after lyophilization was evaluated bygel filtration chromatography (SEC) and ion exchange chromatography(IEC). The assay conditions are as follows.

[Gel Filtration Chromatography]

The samples of the prepared solutions and the lyophilized formulationsafter lyophilization are assayed as solutions prepared by adding aphosphate buffer at pH 7.0 to each sample to contain the humanizedanti-IL-6 receptor antibody in an amount equivalent to about 1 mg/mL.

The samples of the lyophilized formulations on storage are assayed assolutions prepared by adding 0.6 mL/vial of purified water to eachformulation.

The assay solution of each sample is tested under the following assayconditions to determine the component levels (%) of dimers (Dimer) andlow molecular weight degradation products (LMW) in relation to the totalpeak components. In some samples that were found to form aggregateshaving molecular weights higher than those of dimers, the total amountof the aggregates and dimers was reported as dimer level (%).

Assay Conditions

-   -   Column: TSK gel G3000SWxl 7.8 mm I.D.×30 cm (TOSOH).    -   Mobile phase: phosphate buffer, pH 7.0 (50 mmol/L phosphate        buffer, pH7.0, containing 300 mmol/L sodium chloride and 0.05        sodium azide).    -   Sample amount injected: about 60-120 μg expressed as the amount        of the humanized anti-IL-6 receptor antibody.    -   Flow rate: 1 mL/min.    -   Detected at wavelength: 280 nm.        [Ion Exchange Chromatography]

The samples of the prepared solutions and the lyophilized formulationsafter lyophilization are assayed as solutions prepared by addingpurified water to each sample to contain the humanized anti-IL-6receptor antibody in an amount equivalent to about 1 mg/mL.

The samples of the lyophilized formulations on storage are assayed assolutions prepared by adding 0.6 mL/vial of purified water to eachformulation.

The assay solution of each sample is tested under the following assayconditions to determine the component levels of prepeaks (the sum ofpeaks eluted at retention times shorter than that of the majorcomponent) in relation to the total peak components. The prepeaksinclude multiple decomposition products mainly consisting of deamidationproducts of the humanized anti-IL-6 receptor antibody, and low prepeakproduction levels mean that the deamidation of the antibody wasinhibited.

Assay Conditions

-   -   Column: PolyCAT A 10 cm×4.6 mm, particle size 3 μm, pore        diameter 150 nm (PolyLC).    -   Mobile phase: Solution A: 25 mmol/L MES buffer, pH 6.1.    -   Solution B: 25 mmol/L MES buffer, pH 6.1 (containing 250 mM        sodium acetate).    -   Sample amount injected: about 30-120 μg expressed as the amount        of the humanized anti-IL-6 receptor antibody.    -   Flow rate: 1 mL/min    -   Detected at wavelength: 280 nm.

The evaluation results are shown in Table 1. Thus, inhibitory effectsagainst dimer formation could be observed in the samples containingarginine (sample No. 6), histidine (sample No. 7), serine (sample No. 9)and proline (sample No. 10), as shown by obvious reduction in theincrease in dimer level after lyophilization. These effects were higherthan those obtained with sucrose or trehalose known as typicallyoprotectant. All the samples showed almost no low molecular weightdegradation products. In Table 1, Dimer represents dimers, LMWrepresents low molecular weight degradation products, and Pre representsthe sum of peaks eluted at retention times shorter than that of themajor component.

TABLE 1 Dimer (%) LMW (%) MRA Pre(%) Sample Prepared After PreparedAfter Prepared After No. solution lyophilization (Increase) solutionlyophilization solution lyophilization (Increase) 1 0.42 0.58 0.16 0.000.00 17.4 17.0 −0.4  2 0.32 0.54 0.22 0.00 0.05 — — — 3 0.33 0.58 0.250.00 0.00 — — — 4 0.42 1.05 0.63 0.00 0.00 17.6 17.6 0.0 5 0.60 8.377.77 0.00 0.00 17.6 21.3 3.7 6 0.35 0.36 0.01 0.00 0.00 17.0 17.0 0.0 70.70 0.68 −0.02 0.00 0.00 17.0 16.4 −0.6  8 0.33 2.84 2.51 0.00 0.0017.3 18.2 0.9 9 0.33 0.35 0.02 0.00 0.00 17.7 17.7 0.0 10 0.38 0.45 0.070.00 0.00 17.5 17.7 0.2 11 0.35 1.86 1.51 0.00 0.07 — — —

Then, a thermal acceleration test (storage at 40° C. for 1 month) wasperformed on each sample to evaluate the stability of the lyophilizedformulations on storage. The purity of the antibody before and afterthermal acceleration was evaluated by SEC and IEC. The assay conditionsare as described above.

The evaluation results are shown in Table 2. Thus, inhibitory effectsagainst dimer formation could be observed in the samples containingarginine (sample No. 6), histidine (sample No. 7), serine (sample No. 9)and proline (sample No. 10), as shown by the increase in dimer level ofless than 1% after acceleration at 40° C. for one month. All the samplesshowed almost no production of low molecular weight degradationproducts. The samples containing sucrose (sample No. 1), trehalose(sample No. 2), raffinose (sample No. 3), mannitol (sample No. 4),arginine (sample No. 6), histidine (sample No. 7), serine (sample No. 9)and proline (sample No. 10) showed almost no increase in prepeaks by IECanalysis.

TABLE 2 Sample Dimer (%) LMW (%) MRA Pre (%) No. Initial 40° C.-1M(increase) Initial 40° C.-1M Initial 40° C.-1M (increase) 1 0.82 2.922.10 0.00 0.07 17.3 18.3 1.0 2 0.87 4.06 3.19 0.05 0.00 16.8 18.0 1.2 30.96 4.67 3.71 0.00 0.00 16.8 18.8 2.0 4 1.24 4.06 2.82 0.00 0.00 17.418.5 1.1 5 8.72 38.89 30.17 0.00 0.00 21.3 37.5 16.2 6 0.42 0.97 0.550.00 0.00 17.0 17.2 0.2 7 0.72 1.19 0.47 0.00 0.00 16.7 16.8 0.1 8 2.9310.09 7.16 0.00 0.12 17.9 22.6 4.7 9 0.40 1.32 0.92 0.00 0.00 17.8 19.01.2 10 0.56 1.69 1.13 0.00 0.00 17.6 18.8 1.2 11 2.76 18.31 15.55 0.070.00 17.2 23.0 5.8

Example 2 Influence of the Amounts of Arginine and Sucrose Added toLyophilized Formulations on Stability

The influence of the amounts of arginine and sucrose added tolyophilized formulations containing the humanized anti-IL-6 receptorantibody on the stabilization before and after the lyophilizationprocess and on storage was evaluated.

In this study, evaluation samples of sample Nos. 12-17 containingarginine and sucrose each at three levels were prepared. The formulae ofthe prepared solutions of the evaluation samples before lyophilizationare as follows.

TABLE 6 [Formulae of prepared solutions (before lyophilization)] SampleAntibody Sucrose Arginine Polysorbate Phosphate No. mg/mL mg/mL mg/mL 80mg/mL buffer mM pH 12 40 — 25 0.5 15 6.0 13 40 — 17.5 0.5 15 6.0 14 40 —12.5 0.5 15 6.0 15 40 50 — 0.5 15 6.0 16 40 35 — 0.5 15 6.0 17 40 25 —0.5 15 6.0

A glass vial containing 2 mL of the prepared solution of each evaluationsample was lyophilized under the conditions similar to those of Example1 to give a lyophilized formulation. The formulae of the lyophilizedformulations of the evaluations samples are as follows.

TABLE 7 [Formulae of lyophilized formulations (after lyophilization)]Polysorbate Phosphate Sample Antibody Sucrose Arginine 80 buffer No.mg/vial mg/vial mg/vial mg/vial μmol/vial pH 12 80 — 50 1 30 6.0 13 80 —35 1 30 6.0 14 80 — 25 1 30 6.0 15 80 100 — 1 30 6.0 16 80 70 — 1 30 6.017 80 50 — 1 30 6.0

In order to evaluate stability during the lyophilization process, thepurity of each sample of the prepared solutions before lyophilizationand the lyophilized formulations after lyophilization was evaluated bySEC and IEC in the same manner as in Example 1.

The evaluation results are shown in Table 3. Inhibitory effects againstdimer formation could be observed after lyophilization in the samplescontaining arginine at 17.5 mg/mL (sample No. 13) or more. On the otherhand, similar effects could be observed in only the sample containingsucrose at 50 mg/mL (sample No. 14) or more. Thus, it could be foundthat arginine has a better stabilizing effect after lyophilization thansucrose. It should be noted that the molar concentration of sucrose insample No. 15 is equal to the molar concentration of arginine in sampleNo. 12. In the all samples, neither low molecular weight degradationproducts nor increase in prepeaks (IEC) was observed.

TABLE 3 Dimer (%) LMW (%) MRA Pre(%) Sample Prepared After PreparedAfter Prepared After No. solution lyophilization (Increase) solutionlyophilization solution lyophilization (Increase) 12 0.34 0.32 −0.020.00 0.00 16.5 17.1 0.6 13 0.37 0.39 0.02 0.00 0.00 16.6 17.2 0.6 140.36 0.42 0.06 0.00 0.00 16.8 17.2 0.4 15 0.45 0.48 0.03 0.00 0.00 17.117.3 0.2 16 0.48 0.55 0.07 0.00 0.00 17.2 17.8 0.6 17 0.50 0.66 0.160.00 0.00 16.9 17.5 0.6

Then, a thermal acceleration test (storage at 40° C. and 25° C. for 1month) was performed on each sample to evaluate the stability of thelyophilized formulations on storage. The purity of the antibody beforeand after thermal acceleration was evaluated by SEC and IEC in the samemanner as in Example 1.

Evaluation results are shown in Table 4. The inhibitory effects of botharginine and sucrose against dimer formation after acceleration at 40°C. and 25° C. for one month increased as the contents increased.However, arginine showed slightly lower increase in dimer level at lowcontents. At any content, almost no low molecular weight degradationproducts were observed and no increase in prepeaks (IEC) was observed.

TABLE 4 Sample Dimer (%) LMW (%) No. Initial 40° C.-1M (Increase) 25°C.-1M (Increase) Initial 40° C.-1M 25° C.-1M 12 0.31 0.82 0.51 0.47 0.160.00 0.11 0.00 13 0.37 1.15 0.78 0.60 0.23 0.00 0.06 0.00 14 0.41 1.661.25 0.73 0.32 0.00 0.13 0.00 15 0.53 1.07 0.54 0.69 0.16 0.00 0.00 0.0016 0.64 1.63 0.99 0.89 0.25 0.00 0.00 0.00 17 0.70 2.27 1.57 1.12 0.420.00 0.00 0.00 Sample MRA Pre (%) No. Initial 40° C.-1M (Increase) 25°C.-1M (Increase) 12 16.8 16.8 0.0 16.7 −0.1 13 16.7 17.1 0.4 16.8 0.1 1416.7 17.2 0.5 16.7 0.0 15 17.2 17.5 0.3 17.1 −0.1 16 17.1 17.7 0.6 17.10.0 17 17.0 18.0 1.0 17.4 0.4

Example 3 Influence of the pH of Lyophilized Formulations ContainingArginine on Stability

The influence of the pH of lyophilized formulations containing thehumanized anti-IL-6 receptor antibody and arginine on the stabilizationbefore and after the lyophilization process and on storage wasevaluated.

In this study, evaluation samples of sample Nos. 18-21 having aformulation pH in the range of 5.5-7.0 were prepared. The formulae ofthe prepared solutions of the evaluation samples before lyophilizationare as follows.

TABLE 10 [Formulae of prepared solutions (before lyophilization)]Polysorbate Phosphate Sample Antibody Arginine 80 buffer No. mg/mL mg/mLmg/mL mM pH 18 40 25 0.5 15 7.0 19 40 25 0.5 15 6.5 20 40 25 0.5 15 6.021 40 25 0.5 15 5.5

A glass vial containing 2 mL of the prepared solution of each evaluationsample was lyophilized under the conditions similar to those of Example1 to give a lyophilized formulation. The formulae of the lyophilizedformulations of the evaluations samples are as follows.

TABLE 11 [Formulae of lyophilized formulations (after lyophilization)]Polysorbate Phosphate Sample Antibody Arginine 80 buffer No. mg/vialmg/vial mg/vial μmol/vial pH 18 80 50 1 30 7.0 19 80 50 1 30 6.5 20 8050 1 30 6.0 21 80 50 1 30 5.5

In order to evaluate stability during the lyophilization process, thepurity of each sample of the prepared solutions before lyophilizationand the lyophilized formulations after lyophilization was evaluated bySEC and IEC in the same manner as in Example 1.

The evaluation results are shown in Table 5. The dimer levels in theprepared solutions decreased as the pH decreased, but no dimer formationwas observed at any pH after lyophilization. In the all samples, neitherlow molecular weight degradation products nor increase in prepeaks (IEC)was observed.

TABLE 5 Dimer (%) LMW (%) MRA Pre(%) Sample Prepared After PreparedAfter Prepared After No. solution lyophilization (Increase) solutionlyophilization solution lyophilization (Increase) 18 0.42 0.44 0.02 0.000.00 16.5 17.2 0.7 19 0.36 0.35 −0.01 0.00 0.00 16.9 17.3 0.4 20 0.340.32 −0.02 0.00 0.00 16.5 17.1 0.6 21 0.35 0.32 −0.03 0.00 0.00 17.516.6 −0.9

Then, a thermal acceleration test (storage at 40° C. and 25° C. for 1month) was performed on each sample to evaluate the stability of thelyophilized formulations on storage. The purity of the antibody beforeand after thermal acceleration was evaluated by SEC and IEC in the samemanner as in Example 1.

The evaluation results are shown in Table 6. Depending on the dimerlevels of the prepared solutions, the dimer levels before accelerationdecreased as the pH decreased. However, the increase in dimer level wascomparable at any pH after acceleration at 40° C. and 25° C. for onemonth. At any pH, almost no low molecular weight degradation productswere observed and no increase in prepeaks (IEC) was observed.

TABLE 6 Sample Dimer (%) LMW (%) No. Initial 40° C.-1M (Increase) 25°C.-1M (Increase) Initial 40° C.-1M 25° C.-1M 18 0.44 0.95 0.51 0.61 0.170.00 0.00 0.00 19 0.34 0.82 0.48 0.50 0.16 0.00 0.05 0.00 20 0.31 0.820.51 0.47 0.16 0.00 0.11 0.00 21 0.31 0.82 0.51 0.47 0.16 0.00 0.05 0.00Sample MRA Pre (%) No. Initial 40° C.-1M (Increase) 25° C.-1M (Increase)18 17.0 17.2 0.2 17.0 0.0 19 16.8 17.0 0.2 16.9 0.1 20 16.8 16.8 0.016.7 −0.1 21 16.5 16.6 0.1 16.5 0.0

Example 4 Influence of the Antibody Content in Lyophilized FormulationsContaining Arginine on Stability

In order to determine the possibility of enrichment of lyophilizedformulations containing the humanized anti-IL-6 receptor antibody andarginine, the influence of the antibody content on the stabilization ofthe formulations before and after the lyophilization process and onstorage was evaluated.

In this study, evaluation samples of sample Nos. 22-23 containing 80 and240 mg/vial of the antibody in the formulations were prepared. Theformulae of the prepared solutions of the evaluation samples beforelyophilization are as follows.

TABLE 14 [Formulae of prepared solutions (before lyophilization)]Polysorbate Phosphate Sample Antibody Arginine 80 buffer No. mg/mL mg/mLmg/mL mM pH 22 40 25 0.5 15 6.0 23 40 25 0.5 15 6.0

A glass vial containing 2 mL of the prepared solution of sample No. 22or 6 mL of the prepared solution of sample No. 23 was lyophilized underthe following conditions to give a lyophilized formulation.

TABLE 15 Lyophilization conditions Degree of Step Shelf temperaturePeriod vacuum Initial freezing −50° C. ca. 24 hr — Primary drying −20°C. 105 hr 10 Pa  Secondary drying (1)  25° C. 28 hr 6 Pa Secondarydrying (2)  30° C. 10 hr 6 Pa

The formulae of the lyophilized formulations of the evaluations samplesare as follows.

TABLE 16 [Formulae of lyophilized formulations (after lyophilization)]Polysorbate Phosphate Sample Antibody Arginine 80 buffer No. mg/vialmg/vial mg/vial μmol/vial pH 22 80 50 1 30 6.0 23 240 150 3 90 6.0

In order to evaluate stability during the lyophilization process, thepurity of each sample of the prepared solutions before lyophilizationand the lyophilized formulations after lyophilization was evaluated bySEC in the same manner as in Example 1.

The evaluation results are shown in Table 7. At any content, neitherincrease in dimer level after lyophilization nor low molecular weightdegradation products were observed.

TABLE 7 Dimer (%) LMW (%) After After Sample Prepared lyophili- (In-Prepared lyophili- No. solution zation crease) solution zation 22 0.340.32 −0.02 0.00 0.00 23 0.46 0.43 −0.03 0.00 0.00

Then, a thermal acceleration test (storage at 40° C. and 25° C. for 1month) was performed on each sample to evaluate the stability of thelyophilized formulations on storage. The purity of the antibody beforeand after thermal acceleration was evaluated by SEC in the same manneras in Example 1.

The evaluation results are shown in Table 8. The increase in dimer levelwas comparable at any content after acceleration at 40° C. and 25° C.for one month. At any content, almost no low molecular weightdegradation products were observed.

TABLE 8 Sample Dimer (%) LMW (%) No. Initial 40° C.-1M (Increase) 25°C.-1M (Increase) Initial 40° C.-1M 25° C.-1M 22 0.31 0.82 0.51 0.47 0.160.00 0.11 0.00 23 0.43 0.97 0.54 0.61 0.18 0.00 0.00 0.00

Example 5 Selection of the Types and Contents of Fillers on the Basis ofthe Quality of Lyophilized Cakes of Lyophilized Formulations

To determine the influence of filler types on the quality of lyophilizedcakes of lyophilized formulations containing the humanized anti-IL-6receptor antibody, the samples evaluated in Example 1 (sample Nos. 1-11)were visually evaluated for the presence of shrinkage in lyophilizedcakes. Samples showing shrinkage of 1 mm or more in radius inlyophilized cakes were assessed as “shrinkage”.

The results are shown in Table 9. The sample containing no filler showeda shrinking tendency in the lyophilized cake. However, all the samplescontaining fillers except for dextran showed no shrinking tendency inthe lyophilized cakes and good cake forms.

TABLE 9 Filler Phosphate Sample MRA Content Polysorbate buffer No.mg/vial Type (mg/vial) 80 mg/vial μmol/vial pH Cake form 1 80 Sucrose 501 30 7.0 No shrinkage 2 80 Trehalose 50 1 30 7.0 No shrinkage 3 80Raffinose 50 1 30 7.0 No shrinkage 4 80 Mannitol 50 1 30 7.0 Noshrinkage 5 80 Dextran 50 1 30 7.0 Shrinkage 6 80 Arginine 50 1 30 7.0No shrinkage 7 80 Histidine 50 1 30 7.0 No shrinkage 8 80 Glycine 50 130 7.0 No shrinkage 9 80 Serine 50 1 30 7.0 No shrinkage 10 80 Proline50 1 30 7.0 No shrinkage 11 80 — — 1 30 7.0 Shrinkage

Then, the influence of the contents of arginine and sucrose on thequality of lyophilized cakes was evaluated using the samples evaluatedin Example 2 (sample Nos. 12-17).

The results are shown in Table 10. The samples containing 50 mg/vial (25mg/mL in the prepared solutions) or more of arginine or 100 mg/vial (50mg/mL in the prepared solutions) or more of sucrose showed no shrinkingtendency and good lyophilized cake forms.

TABLE 10 Phosphate Sample MRA Sucrose Arginine Polysorbate buffer No.mg/vial mg/vial mg/vial 80 mg/vial μmol/vial pH Cake form 12 80 — 50 130 6.0 No shrinkage 13 80 — 35 1 30 6.0 No shrinkage 14 80 — 25 1 30 6.0No shrinkage 15 80 100 — 1 30 6.0 No shrinkage 16 80 70 — 1 30 6.0 Noshrinkage 17 80 50 — 1 30 6.0 No shrinkage

Example 6 Optimization of the Arginine Content on the Basis of theQuality of Lyophilized Cakes of Lyophilized Formulations ContainingArginine

The influences of the antibody content and the arginine content on thequality of lyophilized cakes of lyophilized formulations containing thehumanized anti-IL-6 receptor antibody and arginine were evaluated.

In this study, evaluation samples of sample Nos. 24-48 containing 40-160mg/vial of the antibody in the formulations were prepared. The formulaeof the prepared solutions of the evaluation samples beforelyophilization are as follows.

TABLE 21 [Formulae of prepared solutions (before lyophilization)]Polysorbate Phosphate Sample Antibody Arginine 80 buffer No. mg/mL mg/mLmg/mL mM pH 24 20 0 0.15 19 6.0 25 20 6.25 0.15 19 6.0 26 20 12.5 0.1519 6.0 27 20 25 0.15 19 6.0 28 20 50 0.15 19 6.0 29 30 0 0.23 19 6.0 3030 6.25 0.23 19 6.0 31 30 12.5 0.23 19 6.0 32 30 25 0.23 19 6.0 33 30 500.23 19 6.0 34, 44 40 0 0.31 19 6.0 35, 45 40 6.25 0.31 19 6.0 36, 46 4012.5 0.31 19 6.0 37, 47 40 25 0.31 19 6.0 38, 48 40 50 0.31 19 6.0 39 600 0.46 19 6.0 40 60 6.25 0.46 19 6.0 41 60 12.5 0.46 19 6.0 42 60 250.46 19 6.0 43 60 50 0.46 19 6.0

A glass vial containing 2 mL each of the prepared solutions of sampleNos. 24-43 or 4 mL each of the prepared solutions of sample Nos. 44-48was lyophilized under the conditions similar to those of Example 1 togive ten vials of each lyophilized formulation. The formulae of thelyophilized formulations of the evaluations samples are as follows.

TABLE 22 [Formulae of lyophilized formulations (after lyophilization)]Polysorbate Phosphate Sample Antibody Arginine 80 buffer No. mg/vialmg/vial mg/vial μmol/vial pH 24 40 0 0.31 38 6.0 25 40 12.5 0.31 38 6.026 40 25 0.31 38 6.0 27 40 50 0.31 38 6.0 28 40 100 0.31 38 6.0 29 60 00.46 38 6.0 30 60 12.5 0.46 38 6.0 31 60 25 0.46 38 6.0 32 60 50 0.46 386.0 33 60 100 0.46 38 6.0 34 80 0 0.61 38 6.0 35 80 12.5 0.61 38 6.0 3680 25 0.61 38 6.0 37 80 50 0.61 38 6.0 38 80 100 0.61 38 6.0 39 120 00.92 38 6.0 40 120 12.5 0.92 38 6.0 41 120 25 0.92 38 6.0 42 120 50 0.9238 6.0 43 120 100 0.92 38 6.0 44 160 0 1.22 77 6.0 45 160 25 1.22 77 6.046 160 50 1.22 77 6.0 47 160 100 1.22 77 6.0 48 160 200 1.22 77 6.0

Ten vials of each sample were visually evaluated for the presence ofshrinkage in lyophilized cakes (by 3 evaluators) in the same manner asin Example 5, and vials showing shrinkage within 1 mm in lyophilizedcakes were assessed as conforming, and the percentage of these vials wasrecorded as percent within limit (PWL). The PWLs of lyophilized cakes ofthe samples are shown as evaluation results in Table 11.

TABLE 11 Sample Antibody Arginin Antibody Arginine PWL No. mg/mL mg/mLmg/vial mg/vial (%) 24 20 0 40 0 0.0 25 20 6.25 40 12.5 0.0 26 20 12.540 25 13.3 27 20 25 40 50 46.7 28 20 50 40 100 0.0 29 30 0 60 0 0.0 3030 6.25 60 12.5 10.0 31 30 12.5 60 25 33.3 32 30 25 60 50 63.3 33 30 5060 100 86.7 34 40 0 80 0 0.0 35 40 6.25 80 12.5 20.0 36 40 12.5 80 2546.7 37 40 25 80 50 100.0 38 40 50 80 100 100.0 39 60 0 120 0 0.0 40 606.25 120 12.5 13.3 41 60 12.5 120 25 53.3 42 60 25 120 50 86.7 43 60 50120 100 80.0 44 40 0 160 0 6.7 45 40 6.25 160 25 16.7 46 40 12.5 160 5053.3 47 40 25 160 100 100.0 48 40 50 160 200 100.0

Samples had substantially no special problem as products if the PWL was30% or more, preferably 40% or more, more preferably 50% or more in theevaluation results.

Sample Nos. 32, 33, 37, 38, 41, 42, 43, 46, 47, and 48 showed goodquality of lyophilized cakes as judged from the proportion of conformingitems free from shrinkage in lyophilized cakes of 50% or more. SampleNos. 33, 37, 38, 42, 43, 47, and 48 showed very good quality oflyophilized cakes as judged from the proportion of conforming items freefrom shrinkage in lyophilized cakes of 80% or more. Thus, the quality oflyophilized cakes increased as the antibody concentration of theprepared solution increased, and the PWL of lyophilized cakes reached50% or more by adding 25 mg/mL or more of arginine when the antibodyconcentration was 30 mg/mL or more. Especially when the antibodyconcentration was 40 mg/mL or more, the PWL of lyophilized cakes reached80% or more by adding 25 mg/mL or more of arginine.

Example 7 Influence of the Types of Fillers on the Viscosity ofReconstituted Solutions of Lyophilized Formulations

The influence of the types of fillers on the viscosity of reconstitutedsolutions of lyophilized formulations containing the humanized anti-IL-6receptor antibody was evaluated.

Reconstituted solutions were prepared by adding 0.6 mL of water forinjection to each lyophilized formulation of sample Nos. 1, 2, 3, 6, 8,9, and 10 among the samples evaluated in Example 1 and used asevaluation samples.

The viscosity of the reconstituted solution of each evaluation samplewas measured by using a cone and plate viscometer. The assay conditionsare as follows.

[Viscometry]

The viscosity of the reconstituted solution was measured by using aviscoelastometer Rheometer AR-1000 (TA Instruments, Inc.).

Assay Conditions

-   -   Geometry: Cone-and-plate system    -   (Angle 2°, Diameter 40 mm, Truncation 61 μm)    -   Test temperature: 25° C.    -   Test cycle:

Steps Period Shear rate Conditioning step 10 sec — Continuous ramp step1 5 min logarithmically increasing from 1 to 300 [1/s] Peak hold step 115 sec constant at 300 [1/s] Continuous ramp step 2 5 minlogarithmically decreasing from 300 to 1 [1/s]

The shear stress imposed on the geometry in Continuous ramp step 1 andContinuous ramp step 2 was monitored and the viscosity in each step wascalculated from an approximate equation for Newtonian fluids. Theaverage of viscosities in Continuous ramp step 1 and Continuous rampstep 2 was reported as the viscosity of each sample.

The results of viscometry on the samples are shown in Table 12.Formulations containing amino acids as fillers showed lower viscositiesthan formulations using sugars as fillers at the same content. Theformulation containing arginine showed a decrease in viscosity of 1mPa·s or more as compared with the formulation containing sucrose.

TABLE 12 Filler Phosphate Sample Antibody Content Polysorbate BufferViscosity No. mg/vial Type mg/vial 80 mg/vial μmol/vial pH mPa · S 1 80Sucrose 50 1 30 7 3.5 2 80 Trehalose 50 1 30 7 3.7 3 80 Raffinose 50 130 7 3.7 6 80 Arginine 50 1 30 7 2.3 8 80 Glycine 50 1 30 7 3 9 80Serine 50 1 30 7 2.7 10 80 Proline 50 1 30 7 2.9

Example 8 Influence of the Antibody Concentration on the Viscosity ofReconstituted Solutions of Lyophilized Formulations Containing Arginine

The influence of the antibody concentration on the viscosity ofreconstituted solutions of lyophilized formulations containing thehumanized anti-IL-6 receptor antibody and arginine was evaluated.

In this study, samples of reconstituted solutions of sample Nos. 49-53having different antibody concentrations were prepared by varying theamount of water for injection added to sample No. 12 among the samplesevaluated in Example 2 as follows. As a reference, a sample of areconstituted solution (sample No. 54) at an antibody concentration ofabout 200 mg/mL was prepared from sample No. 16 evaluated in Example 2consisting of the lyophilized formulation containing the humanizedanti-IL-6 receptor antibody and sucrose and measured for viscosity.Viscometry was performed using the same procedure as described inExample 7.

TABLE 25 Sample Antibody Arginine Sucrose Polysorbate Phosphate No.mg/mL mg/mL mg/mL 80 mg/mL buffer mM pH 49 120 75 0 1.5 45 6.0 50 14087.5 0 1.75 52.5 6.0 51 160 100 0 2 60 6.0 52 180 112.5 0 2.25 67.5 6.053 200 125 0 2.5 75 6.0 54 195 0 171 2.4 73 7.0

The results of viscometry are shown in Table 13. The viscosity increasedwith the antibody concentration, but the viscosity at an antibodyconcentration of 200 mg/mL was 16.9 mPa·s, which was lower than that ofthe reconstituted solution at an antibody concentration of 195 mg/mLcontaining sucrose. Thus, the viscosity of the arginine formulation waslower than that of the sucrose formulation at high antibodyconcentrations despite of the higher molar concentration of arginine,suggesting that arginine may reduce the viscosity of antibody solutions.

TABLE 13 Sample Antibody Arginine Sucrose Viscosity No. mg/mL mg/mL mMmg/mL mM mPa · S 49 120 75 431 0 0 3.4 50 140 87.5 502 0 0 4.9 51 160100 574 0 0 7.2 52 180 112.5 646 0 0 11.0 53 200 125 718 0 0 16.9 54 1950 0 171 500 25.3

The invention claimed is:
 1. An antibody-containing lyophilizedformulation including arginine or a salt thereof, as a lyoprotectant ora filler, wherein the ratio of the total weight of reducing sugars,non-reducing sugars, sugar alcohols and polysaccharides to the antibodyweight is less than 0.5:1, and wherein the content of said arginine orsalt thereof in the formulation is at least 270 moles per mole ofantibody.
 2. The formulation of claim 1 wherein the amino acid is anL-amino acid.
 3. The formulation of claim 1 wherein said ratio is lessthan 0.025:1.
 4. The formulation of claim 1 which is free from reducingsugars, non-reducing sugars, sugar alcohols and polysaccharides.
 5. Theformulation of claim 1 wherein the antibody is a humanized anti-IL-6receptor antibody.
 6. The formulation of claim 1, wherein the ratio ofthe total weight of reducing sugars, non-reducing sugars, sugar alcoholsand polysaccharides to the antibody weight is less than 0.125:1.
 7. Theformulation of claim 1, the ratio of the total weight of reducingsugars, non-reducing sugars, sugar alcohols and polysaccharides to theantibody weight is less than 0.025:1.
 8. The formulation of claim 1,which does not comprise histidine.
 9. The formulation of claim 1,wherein the antibody is an IgG1 antibody.
 10. The formulation of claim5, wherein the antibody is hPM-1.